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Keywords:

  • dynamics;
  • enzyme;
  • flavin;
  • kinetics;
  • P450

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structures of CPR
  5. Transient state spectroscopy of CPR
  6. Extensive conformational landscapes exist in diflavin reductase enzymes
  7. Functional exploration of the conformational landscape in CPR
  8. Concluding remarks
  9. References

There is a mounting body of evidence to suggest that enzyme motions are linked to function, although the design of informative experiments aiming to evaluate how this motion facilitates reaction chemistry is challenging. For the family of diflavin reductase enzymes, typified by cytochrome P450 reductase, accumulating evidence suggests that electron transfer is somehow coupled to large-scale conformational change and that protein motions gate the electron transfer chemistry. These ideas have emerged from a variety of experimental approaches, including structural biology methods (i.e. X-ray crystallography, electron paramagnetic/NMR spectroscopies and solution X-ray scattering) and advanced spectroscopic techniques that have employed the use of variable pressure kinetic methodologies, together with solvent perturbation studies (i.e. ionic strength, deuteration and viscosity). Here, we offer a personal perspective on the importance of motions to electron transfer in the cytochrome P450 reductase family of enzymes, drawing on the detailed insight that can be obtained by combining these multiple structural and biophysical approaches.


Abbreviations:
CPR

cytochrome P450 reductase

CYP

cytochrome P450

ET

electron transfer

MS

methionine synthase

MSR

methionine synthase reductase

NOS

nitric oxide synthase

PELDOR

pulsed electron–electron double resonance

SAXS

small angle X-ray scattering

TUPS

thiouredopyrene-3,6,8-trisulfonate

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structures of CPR
  5. Transient state spectroscopy of CPR
  6. Extensive conformational landscapes exist in diflavin reductase enzymes
  7. Functional exploration of the conformational landscape in CPR
  8. Concluding remarks
  9. References

This review follows from a recent presentation at the 17th International Symposium on Cytochrome P450 Biochemistry, Biophysics and Structure held in Manchester, UK. It presents a perspective from the authors’ viewpoint (and is therefore not intended to be a comprehensive review) of the importance of dynamics in the function of diflavin oxidoreductase enzymes gained from a knowledge of the structures and kinetics of this family of proteins. The emphasis is on cytochrome P450 (CYP) reductases (CPRs) and related enzymes where the integration of structural and functional studies has provided new insight into the concept of ‘functional dynamics’ in this important enzyme family.

A major goal of biophysical research is to establish relationships between protein dynamics and biological function [1–3]. The contributions of motion to the activity of enzyme catalysed reactions or communication networks (protein–protein interactions) are poorly understood. These studies are particularly challenging because motions occur across a variety of time scales, ranging from large-scale domain motion (seconds to milliseconds) to more localized changes in protein structure (microseconds to sub-picoseconds) and bond vibrations (femtoseconds). Evidence, predominantly from studies of protein folding [4] and single molecule dynamics [5], indicates that proteins exist in an equilibrium of conformational states [6]. This equilibrium is defined by the free energy landscape of the protein, comprising many ‘hills and valleys’ representing different protein conformations. This landscape defines the dynamical excursions that proteins make and the challenge is to relate this multidimensional landscape to biological function. Such landscapes are evident in active site closure and opening in many enzyme systems, enabling substrate capture and product release and the positioning of active site residues in the appropriate geometry to enable catalysis [7–11]. Evidence is accumulating (particularly from NMR studies) for a range of dynamic contributions of the protein not only in substrate binding and product release, but also more directly to the enzyme reaction chemistry [12–14]. Landscapes are also central to conformational sampling mechanisms of biological long-range electron transfer (ET). This is apparent in interprotein protein complexes formed by the electron transferring flavoproteins and their multiple protein partners [15,16]. Here, dynamical searches seek out reactive configurations in regions of the landscape that enable productive electronic coupling between redox cofactors. The conformational search required to reach these configurations often limits the rate of the reaction chemistry (i.e. ET) and as such ET is said to be ‘gated’.

There is evidence supporting a role for domain motion in catalysis in the important family of diflavin oxidoreductases typified by human CPR and related family members, including human methionine synthase reductase (MSR) [17], mammalian nitric oxide synthase (NOS) [18–20], and the bacterial proteins sulfite reductase [21] and CYP BM3 [22]. CPR is a membrane-bound NADPH-dependent oxidoreductase that contains FAD and FMN cofactors housed in discrete redox domains separated by a flexible hinge region [23]. CPR catalyses ET from NADPH to CYP enzymes in the endoplasmic reticulum. The chemical mechanism of ET is relatively well understood (although complex) [24–26] and is described in Scheme 1. For the fully oxidized enzyme, NADPH binds to the FAD domain where it transfers a hydride to the N5 of FAD followed by ET from FAD to FMN to yield a quasi-equilibium distribution of two-electron-reduced species (FADH2 FMN, FADH• FMNH• and FAD FMNH2). In the absence of an electron acceptor (such as a cognate CYP mono-oxygenase), a second equivalent of NADPH binds to the FAD domain and transfers a hydride anion to FAD, driving the equilibrium distribution of enzyme states towards the fully (four-electron) reduced species (FADH2 FMNH2). It should be noted that CPR purifies with an air stable FMN semiquinone, meaning that both 2, 4 and 1, 3 electron cycling mechanisms must be considered. A very slow kinetic phase follows four-electron reduction (observed as a slow increase in flavin semiquinone absorbance at 600 nm). This has been attributed previously to the formation of an internal equilibrium between redox states in the absence of an electron acceptor [27]. The activity of CPR is crucial in mediating ET to P450 enzymes in the endoplasmic reticulum. Understanding mechanisms of specificity and redox control in these endoplasmic reticulum redox chains remains crucial to our broader understanding of the metabolism of xenobiotics and drugs, as well as their bioactivation. Below, we discuss the evidence for protein dynamics (in particular redox domain motions) being inextricably linked to redox chemistry in CPR, and we extend this (by analogy and through evidence) to other selected members of the diflavin oxidoreductase family of enzymes.

  • image(Scheme 1.)

[  Simplified reductive half-reaction of human CYP reductase. Adapted with permission from Hay et al. [49]. ]

Structures of CPR

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structures of CPR
  5. Transient state spectroscopy of CPR
  6. Extensive conformational landscapes exist in diflavin reductase enzymes
  7. Functional exploration of the conformational landscape in CPR
  8. Concluding remarks
  9. References

There is strong evidence for conformational variation in CPR arising from rat CPR crystallographic structures. Rat CPR is structurally similar to human CPR (94% sequence identity) [23,28,29]. These structures show several conformational states, ranging from ‘closed’ structures [23] where the dimethylbenzene moieties of the FAD and FMN are juxtaposed, to ‘open’ structures with dramatically increased interflavin distances [28]. Opening and closing of the two flavin binding domains appears to arise via a connecting loop, which acts as a ‘hinge’ (Fig. 1). Beyond the observation of structurally different forms, enzyme variants have been used to more directly access the role of opening and closing of a CPR.

image

Figure 1.  The domain architecture of CPR. The FMN is shown in yellow and the FAD is shown in orange. X-ray crystal structures of rat CPR show different conformational states, ranging from closed structures (left), to more open structures (right) where the FMN moiety is significantly further away from the FAD moiety. The transition between the open and closed structures is proposed to be mediated by a flexible hinge region that lacks electron density in the crystal structures. The open and closed structures are based on Protein Data Bank codes 3ES9 and 1AMO, respectively.

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A CPR variant with four residues deleted from the hinge region (ΔTGEE) was found to have impaired observed rates of interflavin ET but normal observed rates for ET to the CYP redox partner [28]. X-ray crystal structures of the ΔTGEE variant show a significantly extended (open) structure compared to the wild-type enzyme with increased interflavin distances. This is consistent with other studies suggesting that residues required for interaction with CYPs are occluded in closed conformations of CPR [30] but, more importantly, suggests that the reduction of CPR enables the enzyme to adopt a more open conformation. Similarly, deletion of the hinge region gives rise to a dramatic decrease in the rate of interflavin ET [31]. Small angle X-ray scattering (SAXS) suggests that the poor ET rate in the hinge deletion CPR variant may arise from impaired diffusional motion of the FAD and FMN domains, which in turn prevents a favourable domain configuration for interflavin ET [32]. A recent protein engineering study also supports the hypothesis of functionally relevant conformational dynamics in CPR. Here, a non-native disulfide linkage was engineered between the FMN and FAD domains of a wild-type rat CPR [33]. The X-ray crystal structure shows that such a linkage gives rise to an artificially closed structure of CPR with a relatively short interflavin distance. The observed rates of ET from CPR to CYP were found to be significantly impaired in this artificially locked/closed form. However, approximate wild-type levels of reduction of CYP by CPR were rescued upon reduction of the disulfide linkage. These data were rationalized by invoking the need to adopt more open conformations of CPR to interact optimally with partner CYP proteins, consistent with a model where open conformations of CPR may be required to pass electrons to CYPs. However, on distance arguments alone, such an open structure would impair interflavin ET in CPR, suggesting that alternate cycling between open and closed forms of CPR throughout the CPR–CYP catalytic redox cycle is required.

Although X-ray crystallography has provided evidence for the importance of the open conformation of CPR, NMR spectroscopy and SAXS data have provided evidence for the relevance of the closed state [34]. In this case, 15N–1H chemical shift differences were used to monitor variation in the FMN/FAD domain interactions. These studies suggest that CPR can exist as an equilibrium of different conformations. SAXS studies monitoring the scattering profile of both oxidized and four-electron-reduced CPR gave low-resolution molecular envelopes for comparison with the available X-ray crystal structures. These data suggest that the oxidized and reduced states of CPR adopt different conformations, with a more closed form favored on coenzyme binding to both the oxidized and reduced forms. The binding of coenzyme may therefore induce a shift in the equilibrium of conformational states of CPR.

Similarly, in the related diflavin reductase enzyme, NOS, large domain movements are proposed to play an important role in facilitating ET between the various redox centres of the protein [18,20,35]. During the NOS-catalysed reaction, the FMN domain receives electrons from FAD and acts as an electron donor to the oxygenase domain, thereby allowing catalysis by the heme cofactor. However, in the crystal structure of nNOS from rat [20] the enzyme is ‘locked’ in a conformation that prevents ET between the FMN cofactor and the oxygenase domain, which means that catalysis by the active site heme is inhibited. Binding of the activator protein, calmodulin, is proposed to release the FMN domain from its locked position and ensures that the FMN cofactor is able to deliver electrons to the oxygenase domain [18,20,35,36]. This is assumed to involve large-scale conformational movements (approximately 70 Å) of the entire FMN domain and allows the domain to engage in two distinct protein–protein interactions with the FAD and oxygenase domains [18,20,35,36]. These structural studies provide indirect evidence for conformational change being related to ET in the diflavin reductase enzymes. However, several questions arise. For example, is domain motion required for biological function? Also, what is the nature of the conformational landscape? Does the landscape predominantly comprise two states (i.e. open and closed) or is there a more complex distribution giving rise to an ensemble of conformational functional states? Moreover, is domain motion stochastic and uncoupled to the reaction chemistry (a classical ‘conformational sampling’ mechanism) or are motions linked more tightly to the reaction chemistry? Using a variety of kinetic and spectroscopic methods, we have started to address these questions through the integration of several experimental approaches.

Transient state spectroscopy of CPR

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structures of CPR
  5. Transient state spectroscopy of CPR
  6. Extensive conformational landscapes exist in diflavin reductase enzymes
  7. Functional exploration of the conformational landscape in CPR
  8. Concluding remarks
  9. References

Although structural studies can provide detailed information on conformational states, it is more difficult to link these changes to defined mechanistic steps. In CPR, the redox-state dependent absorbance changes of the flavin cofactors allow use of anaerobic stopped-flow techniques to access ET steps [24]. The observed rate constants for formation of two-electron (FMNH FADH) and four-electron (FMNH2 FADH2) reduced CPR can be monitored by following the formation and decay of the di-semiquinoid (FMNH FADH) two-electron-reduced species at 600 nm on mixing with a saturating concentration of NADPH in an anaerobic stopped-flow instrument [27]. The two exponential phases extracted from these reaction traces correspond broadly to the observed rate constants for two-electron and four-electron reduction (termed k1 and k2, respectively). These kinetic phases are shown in Fig. 2 and Scheme 1. There is also a very slow phase observed after four-electron reduction, which is attributed to the formation of an internal equilibrium between redox states in the absence of an electron acceptor [27].

image

Figure 2.  Example stopped-flow absorbance trace for the reduction of CPR with NADPH. The initial two-electron reduction is broadly described by an increase in absorbance at 600 nm, followed by a decrease corresponding to further two-electron reduction to the fully (four-electron) reduced state. After these kinetic phases, there is an additional phase, which has been attributed to the establishment of an internal equilibrium of redox states (EQ). The trace shown is fit to a function with two exponential components: one-exponential for each two-electron reduction.

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However, the direct observation of interflavin ET rates in these diflavin related enzymes is often compromised by the slow rate of electron delivery and the multiple kinetic phases observed in stopped-flow studies [24,27]. Hence, it is important that new methodologies are developed to accurately determine the rates of FAD-to-FMN ET, and to understand how these processes are controlled. The use of laser photoexcitation methods to rapidly inject electrons into redox enzymes represents a powerful tool for studying fast ET reactions in multisite redox enzymes and avoids the mixing limitations imposed by stopped-flow techniques [35–43]. A number of different photoexcitable electron donors have been developed and successfully applied to study catalysis in various redox enzyme systems. Laser excitation of NAD(P)H forms a highly efficient electron donor that has been used to study heme reduction and ligand binding in various CYP enzymes [37,38], and the inter-copper ET in Alcaligenes xylosoxidans nitrite reductase [39,40]. However, because the diflavin reductase enzymes bind NAD(P)H and use it as the natural reductant in catalysis, it has been necessary to search for alternative photoexcitable electron donors.

Initial flash photolysis experiments on CPR involved the excitation of 5-deazariboflavin to generate the deazariboflavin semiquinone, which can then subsequently reduce the FAD cofactor in the protein [41]. However, the quantum yield of the deazariboflavin photochemistry is very low, which means that more efficient photoexcitable electron donors are required if laser techniques are to be useful. One such compound, thiouredopyrene-3,6,8-trisulfonate (TUPS) (Fig. 3), is a strong reductant in its triplet state and can donate electrons to various acceptors [42]. Consequently, TUPS was used to rapidly photoreduce the flavin cofactors in CPR and NOS, which allowed the rate of interflavin ET to be measured in both enzyme systems (Fig. 3) [43]. TUPS can be used both in solution and covalently attached to the protein via a surface cysteine residue and provides a widely applicable method to study the internal ET in these and other multi-cofactor enzymes [43]. A similar approach has also been used for ruthenium polypyridyl complexes, which have been used to study ET from the heme c in cytochrome c to CuA in cytochrome c oxidase and internal ET from CuA to heme a and, subsequently, to the heme a3/CuB binuclear centre, as well as the coupled proton pumping in cytochrome c oxidase [44]. Hence, by using TUPS or ruthenium photochemistry, it should now be possible to identify the key control elements (e.g. amino acid residues, ligands and accessory proteins, such as calmodulin) that regulate the rate of internal ET in the diflavin reductase family [43].

image

Figure 3.  Scheme showing ET from TUPS to the reductase domain of NOS and CPR. Electrons pass from photoexcited TUPS, which can be free in solution or covalently bound to the protein, to the FAD cofactor (red) and onto the FMN cofactor (orange). The structure of NOS reductase is taken from Garcin et al. [20]. Kinetic transients at 630 nm upon laser excitation at 355 nm are shown over 40 μs and 200 ms. Adapted with permission from Heyes et al. [43].

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Overall, the use of various time resolved spectroscopy measurements has demonstrated that interflavin ET in CPR occurs with an observed rate constant in the order of approximately 10 s−1 [24,41,43,45]. Steady-state rate constants using cytochrome c as the electron acceptor similarly fall in the range of approximately 10–100 s−1, although these values reflect a number of first- and second-order steps [25]. A summary of reported rate constants using several different methodologies in different laboratories is given in Table 1, encompassing temperature jump, stopped-flow and laser flash photolysis measurements. These rate constants compare with a theoretical value of approximately 1010 s−1 for free energy optimized ET based on the distance separation (approximately 4 Å) of the redox cofactors observed in the crystallographically defined closed conformation of CPR [46–48]. Such a discrepancy between theoretical ‘intrinsic’ (nonadiabatic) ET rate constants and those measured in the laboratory suggests that there is a chemical and/or conformational gating of the ET chemistry. That is, the ET in CPR is considered to be adiabatic and best described by Eyring theory rather than the nonadiabatic (Marcus) description of ET theory [47,48]. Extensive stopped-flow pH and kinetic isotope effect studies [24], as well as temperature-jump experiments [45], have shown that the internal ET (which establishes the di-semiquinoid two-electron-reduced form in CPR) is not gated by proton transfer. Consequently, it appears more likely that ET in CPR is gated by NADP(H) binding and/or conformational change (see below).

Table 1.   Rate constants for interflavin ET in different redox forms of CPR measured using a variety of kinetic approaches. LFP, laser flash photolysis; T-jump, temperature jump perturbation spectroscopy; NG, not given. Column headings indicate the starting oxidation state.
Experimental methodRate constant for interflavin two-electron-transfer (s−1)
OxidizedOne-electron-reducedTwo-electron-reduced
LFP using 5-deazariboflavin [41]70 ± NG15 ± NG
LFP using TUPS [43]23 ± 3
T-jump [45]55 ± 2
Stopped-flow absorbance [24]21 ± 122.7 ± 0.3

Extensive conformational landscapes exist in diflavin reductase enzymes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structures of CPR
  5. Transient state spectroscopy of CPR
  6. Extensive conformational landscapes exist in diflavin reductase enzymes
  7. Functional exploration of the conformational landscape in CPR
  8. Concluding remarks
  9. References

Transient state kinetic studies have suggested that conformational change gates ET in CPR, although this evidence is based largely on inference from a variety of kinetic experiments rather than by direct observation. Pulsed electron–electron double resonance (PELDOR) spectroscopy has been used to gain insight into the conformational landscape of CPR that likely impacts on conformational gating mechanisms [49]. PELDOR spectroscopy allows access to the broader landscape of enzyme conformations rather than more discrete states accessible by other methods. Using PELDOR techniques, the interflavin distance in the two-electron-reduced (di-semiquinone) form of CPR was monitored. This method offers a way of directly monitoring variation in the conformational state of CPR. The dipole–dipole coupling between the two electronic magnetic moments of the flavin semiquinone centres is related to the distance between them by Eqn (1):

  • image(1)

where g1 and g2 are the g values of the two spins, r is the distance between them, and θ is the angle between the interspin vector and the applied magnetic field. PELDOR results represent the conformational distributions exhibited by the protein just before motion ceased as a result of a lack of thermal energy (samples are frozen at 80 K before measurement). Using this method, the predominant interflavin distance for di-semiquinoid CPR was found to be approximately 36 Å. Binding of NADP+ elicits a redistribution across the conformational landscape with three interflavin distances of approximately 36, 22 and 19 Å, representing three discrete energetic minima (i.e. three distinct conformational substates; Fig. 4A). The distances extracted from PELDOR data agree very well with available open and closed X-ray crystal structures of rat CPR [23,28]. These data point to a conformational free energy landscape that is broad and one that can be remodelled by ligand (e.g. NADP+, 2′-ADP) binding. Furthermore, these data correlate well with NMR data, where coenzyme binding was found to give rise to more closed states of CPR [34]. However, the PELDOR data emphasize that coenzyme binding itself may result in a broad population of CPR conformations, and that the equilibrium distribution is shifted to more closed states. High ionic strength conditions lead to a similar shortening of the interflavin distance. Under these conditions, a continuous broad population of conformations is observed with no discrete minima and allowing for an interflavin distance of approximately 15 Å at closest approach. Taken together, the PELDOR studies suggest that CPR can adopt many conformations with potential implications for ET. That is, shorter interflavin distances will favour ET. Consequently, sampling of conformational space (i.e. motions that transiently populate shorter interflavin distances) may be a mechanism by which CPR controls interflavin ET.

image

Figure 4.  Example data extracted from PELDOR studies, showing the Fourier transforms for both CPR (A) and MSR (B). The upper panels show the apo-enzyme and the lower panels show the NADP+ bound enzyme and, in the case of MSR, the MS-activation domain (AD) bound enzyme. There are a significantly increased number of conformations present in the NADP/AD bound enzyme, and these new conformations display shorter interflavin distances. The structures shown demonstrate the expected conformational transition from open to more closed structures. The MSR structure (grey) includes the FMN domain of CPR (red) and MS-AD (blue) and is for demonstrative purposes only. Adapted with permission from Rigby et al. [17] and Hay et al. [49].

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The related diflavin enzyme MSR is also amenable to PELDOR spectroscopy because the di-semiquinone form of MSR can be stably formed [17]. PELDOR spectroscopy is particularly useful in this case because a full-length crystal structure for MSR is lacking, although structural data are available for the isolated FAD/NADPH-binding domain [50]. As with CPR, the structure is proposed to comprise an FMN domain linked to the FAD domain via a flexible hinge region. MSR catalyses a similar reaction to CPR, where electrons are transferred from NADPH ultimately to the acceptor enzyme methionine synthase (MS) [50]. The PELDOR data suggest that NADP+ binding to MSR induces a broad distribution of conformations with decreased interflavin distances [17] in a similar manner to CPR (Fig. 4B). Furthermore, the PELDOR data suggest that, in the presence of the MSR electron accepting partner MS activation domain, there is also a decrease in interflavin distance. However, the pattern of distances differs from that observed upon NADP+ binding. These data suggest that there is a broad conformational landscape in MSR which is remodelled upon ligand and protein partner binding.

To rule out other sources of an apparent decrease in interflavin distance (such as internal motion of the flavin cofactors), electron nuclear double resonance spectroscopy at multiple temperatures has been used to monitor the hyperfine coupling between atomic nuclei and the unpaired electrons of the flavin semiquinone [17]. These data have allowed conformational variations in the flavin moieties themselves to be assessed. Variation in the hyperfine couplings and line intensities has been attributed to the 8-methyl group dynamics of the flavin semiquinone dimethylbenzene ring alone and, importantly, not the multiple geometries of the flavin moieties bound to the protein. These data therefore provide strong evidence for a broad distribution of conformational states observed in the PELDOR data attributable to domain motion (resulting in more closed conformations), and not the internal motion of the flavin cofactors.

Crystallographic evidence suggests that MSR has an extended hinge region compared to CPR [50]. However, it has been suggested that MSR functions in a similar manner to CPR, with the hinge region allowing toggling between open and closed conformations of MSR. The open conformation is considered necessary for binding of the ET partner protein, MS. Isothermal titration calorimetry has shown that MSR binds to MS with a stoichiometry of 2 : 1 [50]. This implies that only 50% of MSR forms a complex with MS, perhaps consistent with a model in which MSR toggles between several (open and closed) conformations, with only a subset of these conformations interacting with MS. Mechanistically, these multiple conformations may enable direct interaction of the FMN with the FAD in MSR for efficient ET (i.e. the closed conformation), whereas, in the open conformation, endergonic ET to cob(II)alamin (open conformation) in MS is favoured. As such, the extended hinge region may act to ‘swing’ the FMN domain between the two states. There are clear similarities in the models of ET for CPR and MSR, suggesting that conformational sampling mechanisms may be general in the diflavin reductase family.

Functional exploration of the conformational landscape in CPR

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structures of CPR
  5. Transient state spectroscopy of CPR
  6. Extensive conformational landscapes exist in diflavin reductase enzymes
  7. Functional exploration of the conformational landscape in CPR
  8. Concluding remarks
  9. References

Although the PELDOR data provide evidence for a rich conformational landscape in CPR, ideally one would like to assess the importance of conformational change to function (i.e. ET). High pressure can be used to perturb conformational equilibria, as demonstrated previously in studies of enzymatic hydride transfer chemistry [51]. This approach has been extended by using variable pressure, viscosity and ionic strength stopped-flow techniques to perturb the conformational landscape in CPR at the same time as monitoring ET from NADPH to the two flavin moieties [49] (Fig. 5). Solvent-accessible surface area calculations suggest that the CPR water sphere is expected to increase by 100–130 cm3·mol−1 upon moving from a ‘closed’ to ‘open’ conformation [49]. The expectation was that increasing pressure would favour more compact conformations, leading to enhanced interflavin ET, essentially giving rise to enhanced observed rates of two- and four-electron reduction. The effect of pressure on the observed rate constants for ET can be related by Eqn (2) [52]:

  • image(2)
image

Figure 5.  The effect of altering hydrostatic pressure (A), viscosity (B) and ionic strength (C) for two- (k1) and four- (k2) electron reduction in CPR with NADPH. (A) k1 is shown in black and k2 is shown in red. Solid lines show fits to Eqn (2). (B) k1 is shown in red and k2 is shown in blue. Solid lines show fits to Eqn (3). (C) k1 is shown in black and k2 is shown in red. Solid lines show fits to Eqn (4). A key feature of these data is that the observed rate constant for both ET steps shows a significant viscosity, pressure and ionic strength dependence. Adapted with permission from Hay et al. [49].

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where RP = 83.13 cm3·mol−1·bar K−1, k0 is the observed rate constant extrapolated to 0 bar, ΔV is the apparent difference between the volume of the reactant and the transition states and Δβ is the compressibility of the transition state: Δβ = dΔV/dp. The pressure dependence of these observed rate constants k1 and k2 are shown in Fig. 5A. Pressure studies have indicated that both k1 and k2 increase with pressure, consistent with an altered and more functionally favored distribution of conformational states across the energy landscape for interflavin ET at high pressure. Although the pressure data do not provide direct information on the nature of the structural change as a result of perturbing the equilibrium distribution of conformational states (i.e. the energy landscape), the pressure dependence of both k1 and k2 is likely a result of CPR adopting a higher population of closed and ET-competent conformations at high pressure compared to those at 1 bar, as predicted by the surface area calculations. Although there are difficulties in gaining detailed structural insight from the pressure data alone, the pressure-related changes to k1 and k2 indicate a change in the conformational distribution across the energy landscape. These data provide further evidence that conformational sampling is an integral part of the (gated) ET mechanism in CPR.

Our interpretation of data obtained from high pressure studies of ET are also supported by transient state solvent perturbation studies in which k1 and k2 (rate constants for two-elecron and four-electron reduction, respectively) were evaluated as a function of viscosity and ionic strength. The Kramers’ model of the solvent viscosity dependence of unimolecular reactions relates the rate constant to the solvent viscosity through a friction coefficient and is described by Eqn (3) [53]:

  • image(3)

where η is the solution viscosity and σ is the contribution of the protein friction to the total friction of the system. As such, a significant viscosity dependence on both k1 and k2 reflects a significant conformational change associated with ET. The viscosity dependence of these observed rate constants k1 and k2 are shown in Fig. 5B. As the viscosity increases, the rate constants for two- and four-electron reduction decrease significantly (by at least 50%) and express different frictional coefficients with σk1 = 1.5 ± 0.3 and σk2 = 7 ± 6 [49]. The dependence of k1 and k2 on the viscosity of the bulk medium therefore also points to a role for conformational sampling in facilitating ET between the flavins in human CPR.

The ionic strength dependence of a rate constant is derived by combining the Brønsted–Bjerrum and the extended law of Debye–Hückel [54]:

  • image(4)

where I is ionic strength and z is charge. The ionic strength dependence of these observed rate constants k1 and k2 are shown in Fig. 5C. An analysis of the ionic strength dependence of reaction rate indicates there is an increase in k1 and k2 at high ionic strength [49]. Additionally, both k1 and k2 maintain their viscosity dependence at high ionic strength, suggesting that interflavin ET is still conformationally gated in high salt. From the temperature dependence of k1 and k2 [49], increasing the ionic strength lowered the enthalpic barrier to k1 and increased the negative activation entropy. The PELDOR data also suggest that CPR adopts more closed conformations at similarly high ionic strength [49]. These data are then consistent with k1 being (at least partially) gated by motion of the FMN domain, which, under high ionic strength conditions, more favorably redistributes to closed and reactive conformers. The rate enhancement of k2 by increasing ionic strength is not as significant, although it is broadly consistent with the model advanced for k1. These data suggest an overall shielding from net electrostatic charge, which favors conformations that are ‘reactive’ for ET.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structures of CPR
  5. Transient state spectroscopy of CPR
  6. Extensive conformational landscapes exist in diflavin reductase enzymes
  7. Functional exploration of the conformational landscape in CPR
  8. Concluding remarks
  9. References

Results from time-resolved spectroscopy studies in conjunction with the analysis of different types of structural data suggest that the conformational change in CPR is coupled to the ET chemistry. ET is proposed to occur via conformational sampling of redox domains to enhance electronic coupling between cofactors and to guide productive encounters with redox partner proteins (CYPs in the case of CPR; MS in the case of MSR). These variable conformational models have emerged from the integration of structural and biophysical studies. We have recently reported the use of transient state fluorescence resonance energy transfer measurements with CPR [55], providing evidence that opening of CPR is driven by changes in the flavin redox state, giving rise to more open conformations when CPR is reduced. We envisage that such a coupling of ET and enzyme motion supports the vectorial transfer of electrons to cognate monoxygenases (P450 enzymes). As we gain an ever deeper understanding of the relationship between CPR motion and chemistry in vitro, it is important also to consider how these motions might impact on function in vivo. In the cell, CPR is located in the microsomal membrane. There may be an additional layer of detail in the interaction of CPR with cognate CYP monoxygenases that has yet to be resolved. For example, the orientation and translational movement of CPR in the membrane environment may impact on the distribution across the conformational landscape. How these perceived motions relate to issues of specificity in transferring electrons to different CYP partner proteins also needs to be addressed. Ultimately, the relationships between motions, reaction chemistry and the polymorphic variations that contribute, for example, to known developmental abnormalities [56] also need to be analyzed. These are challenging questions, requiring the development of improved biophysical tools, although current knowledge of the importance of motions in CPR and related proteins provides a firm foundation to answer these questions.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Structures of CPR
  5. Transient state spectroscopy of CPR
  6. Extensive conformational landscapes exist in diflavin reductase enzymes
  7. Functional exploration of the conformational landscape in CPR
  8. Concluding remarks
  9. References